PROJECT SUMMARY/ABSTRACT The bladder must refrain from premature contraction during filling and empty when critical intravesical volume and pressure are reached. Abnormal continence or voiding of urine are frequently associated with abnormal excitability of the detrusor smooth muscle (DSM) in the course of bladder filling. Current therapies are largely ineffective and frequently have intolerable side effects. There is a pressing need to better understand the fundamental mechanisms of regulation of bladder function during filling that may yield novel ideas for more efficient control of bladder excitability. Excitatory purines that increase DSM contractility (e.g., ATP and ADP) and inhibitory purines that decrease DSM contractility (e.g., NAD, ADP-ribose, AMP and adenosine) are released from the urothelium and form ?a regulatory purine pool? deep in the bladder wall. The relative composition of this pool (e.g., inhibitory vs. excitatory) might be changing during bladder filling to enable adequate DSM excitability. However, purine-mediated local mechanisms of signaling between the urothelium and DSM during filling are not understood. This project will investigate 1) several mechanisms - release, metabolism and transurothelial transport - that determine the type and relative amount of purine mediators available in suburothelium (SubU)/lamina propria (LP) during filling and 2) influences of extracellular purines on non-neural types of cells in the bladder wall that regulate DSM excitability. Specific Aim 1 will test the hypothesis that asymmetrical availability of purines leads to a higher ratio of inhibitory/excitatory purines in SubU/LP during the storage phase of bladder filling whereas reduction of this ratio at high volume and pressure facilitates micturition. ATP, ADP, NAD, ADP-ribose, AMP and adenosine will be examined simultaneously in SubU/LP and in lumen during filling. Specific Aim 2 will test the hypothesis that metabolism and transurothelial transport of purines regulate adequate purine availability in the SubU/LP during bladder filling. Specific Aim 3 will test the hypothesis that urothelial purines contribute to the intrinsic control of bladder excitability during filling by affecting urothelial cells, submucosal PDGFR?+ cells and DSM cells. To obtain direct access to SubU/LP, we will use a decentralized (ex vivo) bladder model with DSM removed and we will perform in vivo and ex vivo microdialysis of the bladder wall. We will use analytical chemistry, electrophysiology, molecular biology, protein biochemistry, and functional and Ca2+ imaging methodologies, including expression of optogenetic sensors in selected cell types in the bladder wall. Studies will employ transgenic mice such as Pdgfr?egfp/+, smMHC-GCaMP6f, PDGFR?-GCaMP6, Trpv4eGFP, AQP3-GCaMP6m mice and mice with specific gene deletions. Key mechanisms will be validated in bladders from Cynomolgus monkeys (Macaca fascicularis) to determine how knowledge obtained in mouse bladder translates to the primate bladder. At the end of the project period, we will understand the biological significance of urothelial purinergic signaling for mechanosensitive connectivity between the urothelium and DSM and we may identify novel mechanistic targets for the treatment of anomalous bladder excitability.